Congestive heart failure (CHF) typically occurring as a result of myocardial infarction (MI) remains the leading cause of cardiac mortality in the West. Recent CHF treatment research strategies focused on exogenous stem cell administration have been largely disappointing - likely due to poor implant survival and integration into the host myocardium. Cellular reprogramming, allowing the in situ transdifferentiation of cardiac fibroblasts into induced cardiomyocyte-like cells (iCMs), represents a novel myocardial regenerative strategy that may abrogate many challenges of stem cell delivery. However, recent findings that reprogramming factors which consistently induce rodent cell transdifferentiation fail to reprogram human cells suggest that human cells are resistant to reprogramming compared to rodent cells - an important new challenge to this field. We have developed two prototypical ?pro-plasticity? cell reprogramming strategies to test our central hypothesis that these strategies can be used to critically facilitate human cell reprogramming as a means to improve post-MI cardiac function. These pro-plasticity strategies are: 1) transcriptional activation of reprogramming pathways (via p63 downregulation and Hippo pathway override), and 2) induction of a ?trans-cellular? state using an endothelial cell differentiation factor (ETV2 or VEGF) to transdifferentiate fibroblasts into an endothelial cell intermediary. Our corollary hypothesis is that the efficacy of these pro-plasticity strategies can be ascribed to their de-repression of key reprogramming gene activation by epigenetic mediators in higher-order species. The novelty of this work is reflected in the three US patent applications we have filed regarding these discoveries. Our specific aims are accordingly designed to test the hypothesis that each of these two pro-plasticity strategies can enhance human cardio-differentiation and to test a third hypothesis that these strategies can be used to enhance cellular reprogramming and thereby improve post-infarct cardiac function in vivo. In pursuit of these aims, we will use single-cell RNA-seq and ATAC-Seq to identify cardio-differentiating genes and epigenetic factors that are differentially repressed in human vs rodent cells and induced by pro-plasticity strategies and thereby ?reverse engineer? an optimized precision reprogramming cocktail derived from these factors. We will also use CRISPR vs vector-based transgene overexpression strategies to optimize deliver of these reprogramming factors. We will test these strategies in a rat coronary ligation and then a pre-clinical, porcine MI model, testing (AAV- mediated) systemic vs direct myocardial administration strategies. Accomplishment of these aims could redirect efforts in the novel field of cardiac cellular reprogramming and help elucidate a new clinical strategy for treating CHF. 1